The long term goal of the proposed research project is to identify the molecular processes involved in the recovery of renal epithelial cells following injury. The current proposal focuses on two well recognized classes of stress inducible molecular chaperones, heat shock protein 70 and heat shock protein 25 (HSP70 and HSP25) and their function in modulating renal cell injury. The specific aims of this proposal are to determine a) the relative role and interaction of these two classes of heat shock protein as mediators of tolerance to injury, b) their interactions with a panel of proteins involved in maintenance of cellular integrity and polarity (NaK ATPase, ankyrin, spectrin and actin) and c) the molecular processes by which HSP interactions with these integrity related proteins result in restitution of cellular structure and polarity. The generation of transfected heat shock protein is dependent on the vector and is not controlled by the endogenous regulatory mechanisms of the cell. Accordingly, two cell lines which have transfected over expression of HSP70 FLAG (HSP70F) and HSP27 (the human analogue of HSP25) have been established. In the proposed studies the effect of endogenous heat shock proteins (HSP70 and HSP25) on cellular injury will be differentiated from the influence exerted by over expressed heat shock proteins using gene silencing techniques (oligonucleotide decoy and siRNA). The proposed studies will investigate whether these classes of molecular chaperone act autonomously, reciprocally or synergistically to provide protection from injury. To determine the motifs involved in the association between HSP70 and NaK ATPase, as well as the binding affinity of HSP70 for each of the integrity related proteins, immunoprecipitation, protein binding assays and surface plasmon resonance will be utilized. The effect of modulating heat shock protein abundance on the kinetics of restitution of cellular polarity will be explored using immunohistochemistry and biotinylation studies to track movement of displaced NaK ATPase following injury and during recovery. Understanding the dynamics and mediators of acute renal injury and recovery at the cellular level will enable an advanced understanding which will allow the development of therapeutic interventions designed to enhance recovery while avoiding inadvertent exacerbation of injury. The proposed studies will help understand the molecular processes involved in acute kidney failure and recovery. This knowledge will advance and strengthen understanding of the key molecules that protect the kidney from injury and enhance recovery.